From transcriptome to immunome: Identification of DTH inducing proteins from a Phlebotomus ariasi salivary gland cDNA library

Abstract

Delayed-type hypersensitivity (DTH) response to arthropod vector salivary proteins is associated with protection against pathogen transmission. Massive cDNA sequencing, high-throughput DNA plasmid construction and DNA immunisation were used to identify twelve DTH inducing proteins isolated from a Phlebotomus ariasi salivary gland cDNA library. Additionally, nine P. ariasi DNA plasmids produced specific anti-saliva antibodies, four of these showed a Th1 immune response while the other two exhibited a Th2 profile as determined by IgG2a and IgG1 isotype switching, respectively. In order to validate the specificity of sand fly DNA plasmids, mice previously exposed to sand fly saliva were intradermally injected once with selected P. ariasi plasmids and a specific DTH response consisting of infiltration of mononuclear cells in varying proportions was observed at 24 and 48 h. This approach can help to identify DTH inducing proteins that may be related to host protection against vector-borne diseases or other disease agents where cellular immune response is protective.

Introduction

In leishmaniasis, the body of work indicates that a cellular immune response (CIR) to sand fly salivary proteins, likely from CD4⁺ T cells, provides protection against parasite infection [1], [2]. Regardless of the importance of sand fly saliva in Leishmania infection, relatively little information is available on salivary proteins from sand flies other than Phlebotomus papatasi and Lutzomyia longipalpis and, of more importance, on the type of immune responses they generate in the vertebrate host.

The Old World sand fly Phlebotomus ariasi is a vector of Leishmania infantum, the causal agent of visceral leishmaniasis (VL) in humans and dogs in the Mediterranean basin. VL, the most severe form of leishmaniasis, is usually fatal if left untreated. The global incidence of VL is estimated to be 500,000 cases per year [3]. There is no vaccine available for this disease, and the current treatment is based on antimonial drugs with adverse side effects. Recently, the importance of salivary proteins from sand fly vectors as potential targets for vaccine development to control Leishmania infection was put forward [1], [2].

Saliva from sand flies and other blood feeders contains potent pharmacologic components that facilitate blood meals and evading host inflammatory and immune responses [4], [5]. Arthropod vector saliva also plays a role in pathogen transmission. A small amount of vector saliva can exacerbate parasite or virus infectivity [6], [7], [8], [9], [10], [11], [12], [13]. On the other hand, the immune response to arthropod saliva or bites precludes establishment of the pathogen in the vertebrate host [1], [10], [14]. The mechanism of protection may include generation of antibodies that neutralise the effect of the salivary component(s) responsible for pathogen establishment [15]. The vertebrate host CIR against salivary proteins, however, may either kill the parasite or negatively affect its future development by changing the cytokine pattern at the parasite inoculation site. Indeed, Kamhawi et al. [1] reported that animals pre-exposed to P. papatasi sand fly bites generated a strong delayed-type hypersensitivity (DTH) response at the site of the bite that protected them against L. major infection. Moreover, mice vaccinated with a 15-kDa protein of P. papatasi (PpSP15) produced a strong DTH in C57BL/6 mice resulting in protection against L. major infection [2].

Because the CIR to vector salivary proteins depends on the genetic background of the vertebrate host, a broader approach is required for selecting and testing of multiple candidates. Unfortunately, robust algorithms capable of predicting peptides to be presented as MHC class II are not currently available. Such algorithms are required to select transcripts or genes that can generate a cellular immune—particularly a DTH—response.

In the present work, high-throughput approaches based on massive cDNA sequencing, proteomics, and customised computational biology were used to explore the transcripts present in the salivary glands of the sand fly P. ariasi. Transcripts encoding the most abundant secreted proteins were tested by DNA immunisation and reverse antigen screening (RAS) for their ability to induce either cellular or humoral immune responses in animals. The RAS approach consists of a recall immune response produced by the injection of DNA plasmids coding for salivary transcripts in animals previously exposed to sand fly salivary proteins.

High-throughput screening combined with RAS represents a novel method for rapid screening of vector pathogen molecules to search for vaccine candidates based on identification of CIR that can prevent a particular disease.